1. Field of the Invention
The present invention relates to a general process for regenerating cereals, e.g., barley, corn, wheat, rice, sorghum, etc., and to plants produced by the process. More particularly, the invention relates to the use of tissue and cell culture for the regeneration of cereal plantlets from many varieties of cereals.
2. Description of the Prior Art
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation, and for the use of genetic engineering in producing new varieties.
In recent years, plant cell culture successes have had a considerable influence on the respective roles of cell and organism in control of plant growth and development. This concept was supported when isolated plant cells were shown to be amenable to in vitro cultivation and complete plants could be regenerated from cultures derived from somatic cells, either directly via somatic embryogenesis or indirectly via organogenesis. Generally, the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, espectially growth regulators. Early investigations of certain plant species have suggested that exogenous auxin concentration is the major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the formation of organogenesis (shoots, then roots). Many systems that have been developed are at best genus-specific. A few systems with some variations have been found to be applicable to some species of a few genera. However, no prior art system has been found to be generally applicable to most cereals.
One approach which has been tried for several different cereals is the induction of callus on a medium containing a hormone and the regeneration of plants on a medium lacking a hormone. This has been shown by Wernicke et al., Nature 287, 138 (1980) for sorghum using an MS medium and 2,4-dichlorophenoxyacetic acid (2,4-D) as the hormone and leaf tissue as the explant source. This technique has also been used for rice by Nishi et al., Bot. Mag. Tokyo 86, 183 (1973) and Heyser et al., Plant Sci. Letts., 29, 175 (1983). Nishi et al. utilized seed as the explant source, and LS medium containing myo-inositol and thiamine and either 2,4-D, alpha-naphthalene acetic acid (NAA), or indoleacetic acid (IAA) as the hormone. Nabors et al., Planta., 157, 385 (1983) have also used this approach for wheat, rice, oats and millet. Nabors et al. utilized seedling roots as the source of explant tissue, LS medium and 2,4-D or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in combination with kinetin or IAA or tryptophan as the hormone.
Ishii, Proc. 5th Int. Cong. Plant Tissue & Cell Culture, p. 185 (1982) utilized wheat, barley or oat seedlings as the source of explant tissue, MS medium and 2,4-D as the hormone. Similarly, Ozias-Akins et al, Protoplasma 115, 104 (1983) used immature embryo of wheat as the explant tissue, MS or B5 medium and 2,4-D as the hormone. Dale et al., Z. Pflanzenphysiol. 94, 65 (1979) utilized immature embryo of wheat as the explant tissue, B5 medium and 2,4-D as the hormone.
A similar approach which has been utilized to regenerate corn and barley is to use a lower amount of hormone in the regeneration medium than was used in the callus induction medium. This technique has been described by Green et al., Crop Sci. 15, 417 (1975) for corn immature embryos, MS medium and 2,4-D as the hormone. This technique has been described in an abstract of Ching-san et al. for barley immature endosperm, MS medium and an auxin.
A third approach is to induce callus formation and plant regeneration on medium containing different hormones. Masteller et al., Plant Physiol 45, 362 (1970) utilized sorghum shoot tissue as the explant tissue, revised MS medium, a mixture of 2,4-D and coconut milk for callus induction, and a mixture of NAA and coconut milk for plant regeneration. Ram et al., Z. Pflanzenphysiol. 113, 315 (1984) utilized rice seed as the explant tissue, LS medium, a mixture of 2,4-D and kinetin as the hormone for callus induction, and a mixture of IAA and 6-benzyl adenine (BA) as the hormone for plant regeneration. Yan et al., Plant Sci. Letts. 25, 187 (1982) utilized rice leaf blade as the explant tissue, modified MS medium, a mixture of 2,4-D, NAA and IAA as the hormone for callus induction and a mixture of IAA, BA and giberellic acid (GA.sub.3) as the hormone for plant regeneration. McHugen et al., Ann. Bot. 51, 851 (1983) describe the use of wheat node tissue, MS medium, 2,4-D as the hormone for callus induction, and a mixture of IAA and kinetin as the hormone for plant regeneration.
A fourth approach involves the use of at least three medium in sequence for plant regeneration. The media may be used for callus induction, callus maintenance, shoot formation and root formation. An example in rice has been shown by Bajaj et al., Theor. Appl. Genet. 58, 17 (1980). Bajaj et al. utilized rice embryo and MS medium containing yeast extract. 2 mg/l 2,4-D was utilized for callus induction, 1 mg/l 2,4-D for callus maintenance, and IAA and kinetin for plant regeneration. Ahloowalia, Crop Sci. 22, 405 (1982) utilized wheat immature embryos as explant tissue, one-half MS medium, a mixture of 2,4-D, IAA and kinetin for callus induction, a mixture of 2,4-D and zeatin for shoot formation, and NAA for root formation. Sears et al., Crop Sci. 22, 546 (1982) utilized wheat immature embryo as explant tissue, MS medium, 1 mg/l 2,4-D for callus induction, 0.5 mg/l 2,4-D for callus maintenance, 0.1 mg/l 2,4-D for shoot formation, and no hormones for root formation.
This approach has also been described for barley. Orton et al., Theor. Appl. Genet. 57, 89 (1980) utilized immature embryos as explant tissue, modified MS medium containing 5 mg/l 2,4-D for callus induction, B5 medium containing 4 mg/l 2,4-D for callus maintenance, and MS medium containing no hormones for plant regeneration. Orton, Environ. Exp. Bot. 19, 319 (1979) describes a similar process using immature ovaries and B5 medium. The hormones were 2,4-D for callus induction and callus maintenance, and a mixture of GA.sub.3 and kinetin for plant regeneration. Cheng et al., Planta. 123 , 307 (1975) utilized apical meristems as the explant tissue, modified MS and Cheng medium, a mixture of IAA, 2,4-D and 2-isopentyladenine (2-ip) for callus induction, a mixture of IAA, 2-ip, and either 2,4-D, NAA or p-chlorophenoxyacetic acid for callus maintenance, and no hormones for plant regeneration.
In some instances, it is also possible to obtain plant regeneration from callus without transfer from the callus induction medium. Maddock et al., J. Exp. Bot. 34, 915 (1983) described that with certain genotypes of wheat, the medium utilized was suitable for callus induction and plant regeneration from immature embryos. The medium was MS and the hormone was a mixture of 2,4-D and coconut milk. The shoots with roots were cultured on MS medium for proliferation prior to transfer to soil. Kartel et al. describe callus induction and plant regeneration from mature embryos of barley on a B5 medium containing 2,4-D and kinetin.
The prior art does not describe a process for the regeneration of plants from most cereals that is widely applicable and further provides for excellent survival rate and recovery of regenerates. The present invention is the first instance of a broadly and generally applicable procedure for regenerating cereals which has a high survival rate and a high recovery of regenerates.
Cereal plants and seeds are produced by this process. The cereal plants resulting from this process may differ from the starting plant material as a result of somoclonal variation. The pathway is also useful in that it will enable the use of various selection processes to provide further variation. The plants which are produced can be used in conventional breeding programs.